THERAPEUTIC POTENTIAL OF SEAWEED BIOACTIVE …

THERAPEUTIC POTENTIAL OF SEAWEED BIOACTIVE

COMPOUNDS AGAINST Helicobacter pylori

T. VinodhKumar1*, G.Ramanathan2, Henson Jebajose3, B. Ambika4

1 Department of Microbiology AVS College of Arts and Science, Salem, Tamil Nadu, India.

2, 4 Post Graduate Department of Microbiology, Sri Paramakalyani College, Alwarkurichi-

627412.

3 Centre for Marine Science and Technology, Manonmaniam Sundaranar University Rajakkamangalam, Nagercoil, Tamil Nadu, India.

Corresponding Author - T. VinodhKumar1

ABSTRACT

The prevalence of H. pylori is closely tied to socioeconomic conditions and accordingly, this

infection is more common in developing countries than in developed countries such as the

United States. Regardless, it has been estimated that 30–40% of the U.S. population is

infected with H. pylori. Antimicrobial resistance is largely responsible for treatment

failures. Resistance to metronidazole can frequently be overcome by increasing the dose

and duration of treatment with acid suppression. Seaweeds or marine macroalgae are the

renewable living resources which are also used as food, feed and fertilizer in many parts of

the world. Seaweeds are of nutritional interest as they contain low calorie food, but rich in

vitamins, minerals and dietary fibres. In these present investigation metabolites from

seaweeds was evaluated for their antihelicobacterial activity. Two Helicobacter isolates

were found in the samples and have been characterized based on selective biochemical

and morphological characteristics. The isolates were screened for their antibiotics

resistance / sensitivity pattern against twelve commercially availed antibiotics. Among the

antibiotics ten of them exhibit sensitivity to the Helicobacter pylori isolates. But the

isolates showed resistance to Nystatin, omeprazole, metronidazole, ranitidine,

trimethoprim and methicillin. There are eleven marine seaweeds were collected from

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Thondy coastal region ant they were screened for antihelicobacterial activity with crude

metabolites. Among the seaweeds screened against both Helicobacter pylori isolates the

seaweed Padina tetrastomatica (13, 16mm), Halmiola sp (9, 12mm) and Gracilaria edulis

(6, 20mm) exhibit better antihelicobacterial activity in in vitro assay. The mass spectral

data of LC- MS report revealed that the peaks indicated, may be the presence of phenolic

compounds and sulphated polysaccharides which were compared based on earlier reports

on seaweeds.

Keywords – Seaweed, Antibiotics, Antimicrobial resistance, Gracilaria

1.0 INTRODUCTION

Peptic ulcer, lesion that occurs primarily in the mucous membrane of the stomach or

duodenum (the upper segment of the small intestine); Between 10 and 15 percent of the

world’s population suffers from peptic ulcer. Duodenal ulcers, which account for 80 percent of

peptic ulcers, are more common in men than in women, but stomach ulcers affect women more

frequently. The symptoms of gastric and duodenal ulcer are similar and include a gnawing,

burning ache and hunger like pain in the mid-upper abdomen, usually experienced from one to

three hours after meals and several hours after retiring. In the early 1980’s Australian

researcher, Barry Marshall challenged previous theories of ulcer development with evidence

that ulcers could be caused by H. pylori. H. pylori exists the world over and its prevalence in the

population increases with age. In developed countries, prevalence increases about 1% per year

of age where it is rare in children, and reaches 70% in the seventh decade. In developing

countries, more than 50% children acquire the infection by the age of 10 years, and more than

80% of the population gets infected by the age of 20 years. In asymptomatic individuals

prevalence of H. pylori infection varies from 31%-84%.

A recent study of 655 subjects from a teaching hospital in Rome found an overall prevalence of

infection of 40%, with a higher prevalence among nurses and auxiliary employees than among

physicians. Recently, H. pylori were defined as a class 1 carcinogen by the World Health

Organization (Honda et al., 1998). Despite of host’s vigorous immune response, the organisms

evade humoral and cellular immune mechanisms and persist even for decades in the stomach

environment (Baldari et al., 2005). The currently most used eradicating treatment strategies

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for H. pylori infection relies on a triple antibiotic therapy, but the cost of these drugs, poor

patient compliance and emerging of antibiotic-resistant strains are some of the drawbacks of

these therapies. In addition successful eradication therapy does not protect the host from re-

infection (Unge 1999). Vaccination against H. pylori becomes an attractive approach for both

therapeutic and prophylactic purpose. Various antigens which known to be involved in the

pathogenesis of the infection have been proposed as suitable vaccine candidates, such as

urease, the cytotoxin-associated antigen (Cag A), the vacuolating cytotoxin (Vac A), the

Helicobacter pylori adhesion A (Hpa A), and others .The urease which is expressed by almost

all H. pylori isolates is one of the most essential enzymes for virulence and colonization of H.

pylori in the gastric mucosa by neutralizing the microenvironment of H. pylori in the stomach

(Porta et al., 1995). This enzyme consists of two major subunits, UreA (26.5 kD) and UreB (61

kD). The UreB is considered as a reliable vaccine candidate antigen and found to be protective

in mice (Smythies et al., 2005).

The development of safe anti- H. pylori compounds is therefore desirable. Studies have

documented that some medicinal plant extracts have antibacterial activities, including H. pylori

(Cowan, 1999; Isogai et al., 2000; Funtogawa et al., 2004; Ndip et al., 2007). The course of

treatment against helicobacteriosis is usually based on classic triple therapy including proton

pump inhibitors and antibacterial therapy, clarithromycin and amoxicillin. For allergic

patients, amoxicillin could be replaced by metrodinazole. The consumption of foods that

inhibit the growth of bacteria may provide an alternative to current therapies or complement

and expedite current treatments (Keenan et al., 2010). Since seaweeds possess various classes

of chemical constituents with pharmacological importance. The present study was initiated to

screen antihelicobacterial efficacy of seaweeds.

MATERIALS AND METHODS

Sample collection

A total of 5 individuals (2 male; age 50-55years, and 3 female; age 23-45years). Those

individuals who had previously received ulceration symptoms in stomach. Gastric juice as

possible 5 to 10 ml was collected from each individuals during early morning episodes of

intermittent reflux which may facilitate the passage viable organisms into the mouth.

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Bacterial isolates

A total of two clinical isolates of H. pylori obtained from the gastric mucosa of patients. It was

plated on Egg Yolk Emulsion agar medium. Suspicious growth was noted; bacteria were sub

cultured and were incubated for 4 days in a micro aerobic gas environment which was handled

in candle jar method. Bacterial growth was identified as H. pylori on the basis of colony

morphology; positive biochemical reaction for catalase, urease, and oxidase and negative Gram

stain.

Collection of seaweeds

Seaweeds (Gracilaria edulis, Sargassum wightii, Bryopsis sp, Jama sp, Caloroba grapes,

Euchoma sp, Chondrococus sp, Padina tetrastomatica, Caloroba sp, Gracilaria sp, Halmiola sp)

samples which were healthy and fully grown and submerged underwater from the tide pools

were collected from Thondi Coastal region Ramnad District. The samples were washed with

seawater and freshwater to remove salt, epiphytic microorganisms and other suspended

materials. The clean algae were frozen. The dry material was stored.

Extraction of bioactive metabolites from marine algae

Organic solvent methanol was used for extraction. Each powdered sample (5g) was soaked in

about 40 ml of the solvent for three days. The resultant crude extracts were filtered and then

concentrated in a rotatory evaporator at a temperature of less than 400C. The residual water

was removed with a vacuum pump. The crude extracts were weighed and deep frozen (-200C)

until testing.

Analysis of phytochemical constituents

The crude extracts from chosen seaweeds were subjected for the qualitative identify of

different classes of natural compounds, using the methodology of Sofowora (1982). The major

phamaceutically valuable phytochemical compounds viz., alkaloids, flavanoids, steroids,

terpenoids, total sugars, total protein, tannin, phenolic compounds, anthroquinone, and

saponins were investigated in the present study.

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Antibacterial activity of seaweed extract

The helicobacterial activity was determined by well diffusion method. Muller Hinton

Agar (MHA) was (3.1 g/100 ml) weighed and dissolved in 100 ml of distilled water in a sterile

conical flask using test organism. The medium was sterilized by autoclaving and was allowed

to cool at room temperature. The medium was poured into the sterile Petri plate. The

inoculated plates were kept aside for few minutes, using well cutter. Two wells are made in

those plates at required distance. In each step of well cutting, the well cutter was thoroughly

wiped with alcohol, using sterilized micropipette, 100μl of methanol solvents with selected

seaweed extract was added into one well and in another well the same volume of

corresponding controls. The plate was incubated at 37°C for overnight. Inhibition of microbial

growth was determined by measuring the diameters of zone of inhibition.

FTIR spectroscopy analysis

The maximum zone producing seaweeds were selected and it was analyzed by FTIR

Spectroscopy method. The seaweed extract samples (10 mg) was mixed with 100 mg of dried

Potassium bromide (KBr) and compressed to prepare as a salt disc. The disc was then read

spectrometrically. The frequencies of different components present in active sample were

analyzed.

HPLC analysis

Data was obtained at 280 nm by using UV-DAD (UV diode array detector). Data consisted of

various peaks for crude extracts with their respective retention times. Analytical grade HPLC

(high performance liquid chromatography) was performed on a Varian 9021 solvent delivery

system equipped with Varian 9065 Polychrom UV-diode array detector (190-367 nm). Data

was processed by Star Polychrom version 5.2. The system was maintained in a controlled room

temperature at 21±10°C. A flow rate of 1ml/min-1 and injection volume of 10 μl were used.

Sample analysis was performed by gradient elution on a 150 mm x 4.6 mm i.d., 5uM, Luna C-

18(2) column (Phenomenex, Australia) with guard column (Phenomenex, Australia). The

mobile phases were freshly prepared and degassed under vacuum using Phenomenex nylon 45

μm membranes and sonicated in a sanophon ultrasonic bath (Ultrasonic Industries Pty. Ltd,

Sydney, Australia) for 15 minutes prior to HPLC analysis.

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LC-MS (Liquid chromatography mass spectrometry)

Liquid chromatography–mass spectrometry (LC–MS) of the methanolic extracts of selected

seaweed species was performed on a Micromass Quattro micro tandem quadrupole mass

spectrometer (Waters, Manchester, UK). LC separation was attained by a Waters liquid

chromatograph (Waters, Milford, USA), consisting of a 2695 Separation Module and 2487 dual

wavelength UV detector operated at 240 and 280 nm. Columns and gradients were same as

described previously for analytical scale HPLC. An injection volume of 10 μL and a constant

flow of 1 ml/min-1 were used for each analysis. The entire flow from the LC was directed into

the mass spectrometer. Data was acquired by the Masslynx data system for both the MS and

UV data.

Molecular mass from LC-MS data

LC-MS chromatograms obtained for two crude extracts were analysed by Masslynx to get

spectra of various peaks. Spectra were recorded in negative and positive ion mode to estimate

molecular masses of various components. Only major peaks in each chromatogram were

analyzed for determination of molecular masses. Determination of molecular mass from LC-MS

data can be a useful tool to identify components on the basis of molecular masses.

RESULT & DISCUSSION

In this present investigation there are two Helicobacter isolates retrieved from five

patients with the Age group of 23-53 was analyzed. Among them 3 female and 2 male all are

susceptible to ulcer symptoms of intermitted reflux. The isolates were subjected for to screen

against commercially availed antibiotics to evaluate their sensitivity / resistant pattern against

12 antibiotics. Among the two strains adopted, the bacterial isolates 2, was more susceptible

than isolate 1. Helicobacter isolate 1 was resistance to Nystatin, Omeprozole, Metronodazole

and Ranitidine. Su et al., (2013) revealed that the resistance rates to clarithromycin,

metronidazole, levofloxacin, amoxicillin, gentamicin and furazolidone were 21.5, 95.4, 20.6,

0.1, 0.1 and 0.1%, respectively. Double, triple and quadruple antibacterial resistant

percentages were 25.5, 7.5 and 0.1%, respectively. A positive association between the

resistance to levofloxacin and to clarithromycin was found, but there was a negative

correlation in the resistances to levofloxacin and to metronidazole.

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Antihelicobacterial efficacy was accessed by crude seaweed extracts, which was

sourced from Thondy coastal region against Helicobacter isolates 1 and 2. It was found that,

among the seaweed extract screened, the seaweeds Gracilaria, Padina and Halmolia exhibit

better antihelicobacterial activity. Among the Helicobacter isolates, the isolate 2 was more

susceptible with seaweed extract. Time kill assay was also performed; it was found that, higher

concentration of seaweed extract of both Padina tetrastomatica and Gracilaria edulis control

the multiplication of the pathogen with increased concentration at 12 hours.

Antihelicobacterial efficacy was accessed by crude seaweed extracts, which was

sourced from Thondy coastal region against Helicobacter isolates 1 and 2. It was found that,

among the seaweed extract screened, the seaweeds Gracilaria, Padina and Halmolia exhibit

better antihelicobacterial activity. Among the Helicobacter isolates, the isolate 2 was more

susceptible with seaweed extract. Time kill assay was also performed; it was found that, higher

concentration of seaweed extract of both Padina tetrastomatica and Gracilaria edulis control

the multiplication of the pathogen with increased concentration at 12 hours.

The potential seaweed extracts which shows significant helicobacterial efficacy was

further evaluated to characterize the active metabolites by FTIR, HPLC and LC-MS analysis

based on that it was indicated that the active principles may be phenolic, sulphated

polysaccharides which is present as major phytochemical constituents in seaweeds. Time hill

assay was performed against H. pylori isolated by using potential seaweed metabolites with 1x,

4x, and 16 x concentrations for 12 hours. Efficiency of reduction in bacterial population was

collected in log CFU for every 3hours incubation. It was found that 16 x concentrations of all

the four seaweed extracts reduced the population considerably. FTIR analysis was followed to

the potential seaweed extract. The peak value reveals the stretching point of the particular

compound which is present in the seaweed extract. Gracilaria edulis have 29 peaks and Padina

tetrastomactica have 31 peaks

Retention times of all the compounds which are presented in the extracts were

detected. The identification of the peaks is based on the analysis of their retention time. The

Retention time of identified peaks for Gracilaria edulis are 1.400, 2.183, 2.303, and 2.753 and

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for Padina tetrastomatica is 1.427, which indicates the presence of halogenated / sulfated

polysaccharides based on earlier reports on seaweed bioactive metabolites. The peak value of

Gracilaria edulis and Padina tetrastomatica were analyzed and showed in. The major peaks

obtained at four different alternative scans. The mass spectral data revealed that the peaks

indicated, may be the presence of phenolic compounds and sulphated polysaccharides which

were compared based on earlier reports on seaweeds.

Padina tetrastomatica Gracilaria edulis

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Conclusion

In humans, Helicobacter pylori colonizes the stomachs of about half of the world’s

population and highly associated with the number of most important disease of the upper

gastrointestinal tract particularly prevalence in developing countries. Eradication of the

H. pylori primarily dependent on continued application of antibiotics. Although effective,

repeated use of these chemical drugs have sometimes resulted in the development of

resistance and had undesirable effects. These problems have highlighted the need for the

development of new strategies for selective H. pylori eradication. Marine plants may be an

alternative source of materials for the H. pylori eradication because they constitute a rich

source of bioactive chemicals. The present study also found that the seaweeds Gracilaria

edulis, Padina tetrastomatica showed antihelicobacterial activity. The importance of

seaweed for human consumption is well known in many countries, they offer a good source

of recovery of various useful chemicals particularly polysaccharide present in most of the

seaweeds may used for the treatment of intestinal and stomach disorders. In this present

investigation Padina tetrastomatica has proven antihelicobacterial activity based on the

outcome of this investigation the active metabolites derived from seaweeds may be an

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alternative therapeutic agent for the treatment of Helicobacter infection but still further

evaluation and complete structural characterization of active metabolites is warranted

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